Down‐regulation of SARS‐CoV‐2 neutralizing antibodies in vaccinated smokers

Vaccination is an effective approach to help control coronavirus disease 2019 (COVID-19). However, since the vaccines produce a heterogenous immune response, the risk of breakthrough infection is increased in vaccinated individuals who generate low levels of neutralizing antibodies (NAbs). It is therefore paramount in the fight against COVID-19 to identify individuals who have a higher risk of breakthrough infection despite being vaccinated. Here we addressed the effect of cigarette smoking on the production of neutralizing antibodies (NAbs) following COVID-19 vaccination since smoking profoundly suppresses the adaptive immune response to pathogen infection and the association between vaccination and smoking remains unclear. The SARS-CoV-2 Spike antibodies and NAbs (days 0, 14, 42, and 90) were measured in 164 participants received two vaccine doses of an inactivated vaccine (Sinovac-CoronaVac) longitudinally. Anti-Spike antibodies was elevated 14 and 42 days after COVID-19 vaccination compared to baseline (i.e., “Day 0”). Notably, RBD antibodies showed significantly higher expression in the nonsmoking group (n=153) than the smoking (n=11) group on day 42 (p<0.0001, Student’s t-test). NAbs continually increased after the first and second vaccine dose, peaking on day 42. NAbs titers then significantly decreased until day 90. Compared to nonsmokers, the NAb levels in smokers remained low throughout the period of testing. The median NAb titers in the smoking group was 1.40-, 1.32-, or 3.00-fold lower than that of nonsmoking group on day 14, 42, or 90, respectively. Altogether, our results indicate that smoking is a specific risk factor for COVID-19 breakthrough infection following vaccination.

"Day 14″"), and their serum was collected longitudinally on days 0, 14, 42, and 90. Since the SARS-CoV-2 S protein is required for viral entry, serological antibodies on days 0, 14, and 42 targeting S protein and its specific domains, including subunit 1 (S1), receptor binding domain (S-RBD), and subunit 2 extracellular domain (S2ECD), were detected using a protein array (Supporting information). Briefly, the array spotted with these S truncated proteins was incubated with serum for 30 min at room temperature. The serum antibodies bound to their target proteins on array can be visualized by a Cy3 Affinipure donkey anti-human IgG(H+L) antibody and a microarray scanner. Prior to the data analysis, the Z-score was calculated using the fluorescent signal intensity of the protein array as previously described. 7 As shown in Figure 1, antibody levels to S protein and domains (S1, S2ECD, S-RBD) were elevated 14 and 42 days after COVID-19 vaccination compared to baseline in both participant groups (i.e., "Day 0″"). The expression of these antibodies was elevated faster in the nonsmoking group (n = 153) than the smoking (n = 11) group, in which the S-RBD antibodies (p < 0.0001, Student's t-test) showed the significant differentiation on day 42 (p < 0.0001, Student's t-test) ( Figure 1A,B, Figure S1 and S2).
The SARS-CoV-2 Spike D614G substitution is prevalent, with data showing that it increases SARS-CoV-2 infectivity, competitive fitness, and transmission in primary human cells. 8 Therefore, we measured the levels of NAbs on days 0, 14, 42, and 90 following the first vaccine dose using a SARS-CoV-2 pseudovirus neutralization assay with the D614G substitution (Table S3 and Supporting information). 9 NAbs continually increased after the first and second vaccine doses, peaking on day 42, and then significantly decreased until day 90 ( Figure 1C).
We then compared the relative changes (ratio) of anti-RBD antibodies and NAbs longitudinally (0, 14, 48, and 90 days) between nonsmoking and smoking groups. The results indicated that there was no significant difference between smoking and nonsmoking groups from 0  NAb expression in nonsmokers and smokers using a SARS-CoV-2 Omicron pseudovirus neutralization assay, respectively. The color represents and vaccinated individuals in each nonsmoking and smoking group. NT50 represents the antibody titer that resulted in 50% pseudovirus neutralization (pNT50). *** represents the significance with a p-value less than 0.01 to 14 days ( Figure 1D). However, the expression of anti-RBD antibodies in the smoking group was significantly lower than that of the nonsmoking group after 14 days. Whereas the NAbs in the smoking group was significantly lower than that of the nonsmoking group after 42 days, demonstrating the specificity of cigarette to suppress NAb production through regulating humoral immunity ( Figure S3). Notably, the median NAb titers in the smoking group was 1.40-, 1.31-, or 3.00-fold lower than that of the nonsmoking group on days 14, 42, or 90, respectively ( Figure 1E). No correlation was observed between NAbs and other factors [(i.e., age, sex, body-mass index (BMI)] ( Figure S4). As the evolution of SARS-CoV-2 viral sequence, the new Omicron (B.1.1.529) and its subvariants (BA.2, BA.3, BA.4, and BA.5) continually emerged, spread and has become the predominant variant in circulation around the world. In order to know the association between smoking and NAbs to Omicron, we tested the responses of NAbs to Omicron (BA.1.) in the smoking (n = 8) and the nonsmoking group (n = 8) using pseudo virus assay and the serum samples on 42 days after second vaccination ( Figure 1F, G). The results showed that the expression of NAbs was significantly decreased in both smoking and nonsmoking groups. In the smoking group, 62.5% (5/8) vaccinated individuals were below the detection limit (NT50 = 10). Comparably, 37.5% (3/8) vaccinated individuals were below the detection limit (NT50 = 10) in the nonsmoking group. The results confirm the potential influence of smoking on the NAb expression.
There have three limitations in this study. First, the number of clinical samples was limited. The results should be validated in a large different population in future. Second, the dose of cigarette in smoking participants on the suppression of immunity was not investigated due to the unavailability of clinical samples. Third, as the new variants (Omicron, BA.5.) with high infection capability continue to appear, and the relationship between the smoking and the immunity to these newly emerged SARS-CoV-2 variants should be investigated as well.
Altogether, our results demonstrate the capability of smoking to suppress NAb expression 14 days after inactivated vaccination, which may serve as a specific risk factor for COVID-19 breakthrough infection following vaccination. Further investigation of smoking and how it affects NAb levels in response to COVID-19 vaccination with a larger patient cohort and other COVID-19 vaccines is warranted.

A C K N O W L E D G M E N T S
We thank all vaccine recipients for providing serum samples. We also thank Dr. Brianne Petritis for her critical review and editing of this paper. This study is supported by

C O N F L I C T O F I N T E R E S T
The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

A U T H O R S ' C O N T R I B U T I O N
Xiaobo Yu and Shubin Guo designed the study. Fei Teng collected the vaccine recipients' serum samples and clinical data. Wang Hongye, Zhang Xiaomei, and Liang Te collected the experimental data. Jiahui Zhang and Xiaobo Yu executed the statistical analysis, and visualization. Xiaobo Yu wrote the manuscript.

E T H I C S A P P R O VA L
This study was approved by the Institutional Review Board and Medical Ethics Committee of Beijing Chaoyang Hospital (Ethical approval number: 2020-ke-491). Each recipient signed an informed consent.

D ATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are included in this published article and its supplementary information files.